1. Field of the Invention
The invention relates to novel nucleic acids and polypeptides encoded thereby, whose expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain bather functionality. These polypeptides are designated herein as “lipopolysaccharide-sensitive” polypeptides (LPSS polypeptides). The invention further relates to methods useful for controlling blood-brain barrier properties in mammals in need of such biological effects. This includes the diagnosis and treatment of disturbances in the blood-brain/retina barrier, brain (including the eye) disorders, as well as peripheral vascular disorders. Additionally, the present invention further relates to the use of anti-LPSS polypeptide antibodies or ligands as diagnostic probes, as blood-brain barrier targeting agents or as therapeutic agents as well as the use of ligands or modulators of expression, activation or bioactivity of LPSS polypeptides as diagnostic probes, therapeutic agents or drug delivery enhancers.
2. Description of the Background Art
In order to function properly, neurons require a tightly regulated extracellular milieu. This essential, well-defined microenvironment is locally maintained by nursing brain cells called astrocytes (or astroglia). To cope with the considerable and variable dissimilarity between the composition of the blood and the extracellular compartment of the brain, the central nervous system (CNS) is also shielded from the general blood circulation by a number of blood-CNS barriers, i.e. the blood-brain barrier, blood-cerebral spinal fluid (CSF) barrier, pial vessel-CSF barrier, the ependyma and glia limitans, and also the blood-retina barrier, blood-nerve barrier, blood-spinal cord barrier. The blood-brain barrier (BBB) is considered as the most important blood-CNS barrier, because it covers a 1000 times larger surface area when compared to the other blood-CNS barriers. The BBB is characterized by a unique tight endothelial cell layer that covers capillary blood vessels in the CNS. Again, astrocytes are the principal inducers of BBB properties in these endothelial cells, by projecting ‘glial foot’ on the capillaries.
In particular, the BBB regulates the trafficking of ions (Na+, K+, Ca2+), water, nutrients, metabolites, neurotransmitters (glutamic acid, tryptophan), plasma proteins (albumin, fibrinogen, immunoglobulins), cells from the immune system and also xenobiotics (drugs) in and out of the brain. The capillary endothelium in the brain has special properties when compared to peripheral capillaries. It has narrow tight-junctions, no fenestrae, low pinocytotic activity and a continuous basement membrane. The narrow tight-junctions result in a high electrical resistance of 1500-2000 Ohm·cm2. In addition, the endothelial cells have a negative surface charge that repulses negatively charged compounds. They have many mitochondria and enzymes to break down compounds and various selective transport systems to actively transport nutrients and other compounds into and out of the brain. Under healthy conditions, the BBB not only regulates the entry of drugs or endogenous compounds into the brain, but also cellular infiltration is lower compared to peripheral organs. The normal endothelial cell layer provides a thrombo-resistant surface that prevents platelet and leukocyte adhesion and activation of any coagulation system. The highly specialized brain microvascular endothelial cells form a tight barrier which isolates the brain from immune surveillance, and allow only a few mononuclear cells (such as activated T-cells) to migrate into the CNS. The low expression of major histocompatibility complex antigens, the low number of antigen-presenting cells in the healthy CNS, and the fact that the CNS is not properly drained by a fully developed lymphatic vasculature, make the brain an “immuno-secluded” site.
The present understanding of the anatomical basis of the BBB is that it functions as a dynamically regulated organ, influenced by peripheral (e.g., cortisol, adrenaline) and local (e.g., cytokines, chemokines) hormones. In addition to astrocytes, several other cells like pericytes, neurons and cells of the immune system, influence its properties. Next to that, the endothelium is involved in other processes like coagulation, control of vasotonus, antigen-presentation and the control of the basement membrane by e.g., growth factors. Particularly, under pathological conditions like brain and cerebral inflammation, angiogenesis in brain tumors, the activated endothelium plays an important role.
In general, the BBB can be regarded as an organ that serves to protect the homeostasis of the brain. Not surprisingly, dysfunction of the BBB plays a central role in the vast majority of brain disorders. Some examples are:    i. Cerebral vasogenic edema is the result of disease (inflammation) induced leakage of plasma proteins and water from the blood into brain tissue. This is the principal cause of death and disabilities in disorders like stroke, cerebral infections, head trauma, brain tumors and multiple sclerosis. The edema causes the brain to swell within the rigid environment of the scull. The resulting elevation in intracranial pressure may subsequently lead to herniation of the brain followed by failure of essential brain functions like respiration and, if left untreated, results in severe disabilities, coma and even death.    ii. In multiple sclerosis, activated autoreactive T cells cross the activated BBB. Within the CNS, these T cells induce an inflammatory response targeted against myelin, which also causes a disruption of the BBB. Autoantibodies and complement factors now cross the disrupted BBB, which leads to the process of demyelination. Now, myelin fragments also leak back into the periphery through the disrupted BBB, where it activates more autoreactive T cells and increases the production of more autoantibodies.    iii. Failure to secure the delicate ion and neurotransmitter balances within the extracellular fluid leads to impaired neuronal signaling and therefore to impaired cognitive functioning, neuropsychiatric disorders or epileptic seizures.    iv. Impaired clearance of toxic proteins across the BBB into the blood stream has recently been linked to the pathogenesis of neurodegenerative disorders like Alzheimer's disease and prion diseases like Creutzfeldt-Jakob disease and BSE. Pathological accumulation of such proteins leads to neuronal cell death and subsequently to impaired cognitive functioning.
Healing a dysfunctional BBB thus opens new avenues for the treatment of brain disorders. Brain disorders are the principal cause of morbidity and disabilities in the western world. The identification and characterization of novel LPSS polypeptides, whose gene expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain bather functionality, will prove useful to meet these needs.
In addition to the desirable drugs with a BBB-healing capacity for the treatment of brain disorders, a proper functioning BBB is also essential to block or reduce the entry into the brain of lymphocytes, which mediate an immune response. The same holds for the entry into the brain of metastatic cancer cells. The identification and characterization of novel LPSS polypeptides, whose gene expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain barrier functionality, will prove useful to meet these needs.
The BBB, however, also limits the delivery of xenobiotics (such as drugs and diagnostic agents) to the brain, which complicates classical drug therapy (i.e. targeted against neurons) of brain disorders. It is therefore also desirable to either manipulate the permeability of the BBB in order to deliver blood-borne, membrane-impermeant drugs to the brain by reversibly opening the BBB, or to selectively target drugs to the brain via endogenous BBB transport systems. The same holds for drug delivery across the blood-testis barrier and the blood-placenta barrier. The identification and characterization of novel LPSS polypeptides, whose gene expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain barrier functionality, will also prove useful to meet these needs.
It is also desirable to manipulate BBB properties in microvessels of other organs than the brain or eye affected in vascular disorders. Introducing BBB properties in peripheral microvessels will be beneficial in conditions involving (micro)angiopathies, pathological angiogenesis, failure of blood-testis barrier or blood-placenta barrier, and conditions such as pulmonary edema, shock caused by bacterial endotoxins, hyperfibrinolysis and anaphylactic shock. The identification and characterization of novel LPSS polypeptides, whose gene expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain barrier functionality, will also prove useful to meet these needs.
It has been known for a long time that brain astrocytes induce BBB properties in brain capillary endothelial cells (BCEC) by the projection perivascular end feet (Arthur et al., 1987, Brain Res. 433: 155-159; Janzer and Raff, 1987, Nature 325: 253-257). It is also known for a long time that this induction is brought about by soluble factor(s), since astrocyte conditioned medium (ACM) can reproduce some of the inductive effects (Tio et al., 1990, Eur. J. Morphol. 28(2-4): 289-300). Several candidate molecules have been identified, capable of mimicking aspects of ACM-mediated barrier induction in BCEC; these include TGFβ, GDNF, bFGF, IL-6 and steroids. Others have found that the factor is not a protein or peptide and that it contains an iron-nitric oxide adduct (Federici et al., 1995, J. Neurochem. 64(3): 1008-1015; Regina et al., 2001, Biochim Biophys. Acta 1540(3): 233-242). So one can conclude that despite the effort of several research groups, the responsible astrocyte-derived factor has not been identified yet.
In previous experiments, we found that primary isolated BCEC, exposed to ACM, retained many of the essential BBB properties in culture (Gaillard et al., 2001, Eur J Pharm Sci. 12:215-22). By introducing primary cultured brain astrocytes at the bottom of the cell culture well, the transendothelial electrical resistance (TEER) across BCEC monolayers cultured on filter inserts above was increased to about 150% of BCEC monolayers cultured in ACM alone. Moreover, when culturing the astrocytes on the bottom side of the filter insert, thus in close proximity of the BCEC, TEER multiplied by a factor 3-8. In addition, the paracellular transport of sodium fluorescein (FLU, mol. weight 376 Da) and FITC-labeled dextran (FD4, MW 4 kDa) decreased to about 50% of BCEC cultured in ACM alone. In conclusion, the proximity of astrocytes from the BCEC determined the magnitude of the effect, although they did not make physical contact with the BCEC (Gaillard et al., supra).
TEER is a sensitive measure to quantify the permeability of small ions through the tight junctions between BCEC. TEER thus represents the functionality of tight junctions, which are considered the major hallmark of the BBB. The absolute value of TEER is mainly dependent on the amount and complexity of tight junctions between the cells. Likewise, this is also the limiting factor for the paracellular transport of large and hydrophilic compounds.
In additional studies, we found that astrocytes cultured on the bottom side of the filter insert:
(1) maintained (or re-induced) the expression of P-glycoprotein (Pgp, a drug efflux pump involved in multidrug resistance) on BCEC after the first passage (Gaillard et al., 2000, Pharm. Res. 17:1198-1205);
(2) decreased the sensitivity for vinblastine induced BBB disruption (a Pgp functionality assay) (Gaillard et al., 2000, supra);
(3) induced active transport of Pgp substrates from the basolateral (CNS) side to the apical (blood) side of a filter, while this was not observed in BCEC monolayers, despite the fact that Pgp was expressed in BCEC monolayers (Gaillard et al., 2000, supra);
(4) mediated a protective response to LPS-induced BCEC disruption (Gaillard, P., 2000a, Ph.D. Thesis, Leiden University, p 81-97). None of these effects were induced by ACM alone. Apparently, the physical and proximate presence of astrocytes on the bottom side of the filter inserts is superior in inducing BBB properties in BCEC when compared to ACM alone.
There exists thus a need for additional products, methods and assays that provide a means to control BBB properties or identify and modulate cellular responses to early dynamic inflammation induced changes in BBB functionality and tissue response to such changes. Such products, methods and assays will provide benefit in numerous medical conditions and procedures, such as those mentioned above.